Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability

Abstract

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture. PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0230253 March 16, 2020 1 / 18 OPEN ACCESS Citation: Struijk RB, Dorssers LCJ, Henneman P, Rijlaarsdam MA, Venema A, Jongejan A, et al. (2020) Comparing genome-scale DNA methylation and CNV marks between adult human cultured ITGA6+ testicular cells and seminomas to assess in vitro genomic stability. PLoS ONE 15(3): e0230253. https://doi.org/10.

ITGA6+ Human Testicular Cell Populations Acquire a Mesenchymal Rather than Germ Cell Transcriptional Signature during Long-Term Culture

“…The chosen time points reflect distinct morphological changes of the PTC culture. During culture, we observed a notable transition from mostly floating, round cells at the onset of culture to a pronounced monolayer of attached spindle-shaped cells from day 13 onwards, with a smaller population of round germ cells adhering to the monolayer (Figure 1A, Video S1), consistent with previous studies that have reported similar transitions [21,22,29,35]. Throughout culture, PTCs were enriched for SSCs through MACS sorting for ITGA6+ cells, resulting in SSC-enriched PTC fractions (hereafter termed ITGA6 + PTC) obtained at six culture time points from four unique testicular tissue donors (total of 24 fractions) (Figure 1B).…”

“…In that study, the expression of PDGFRA1 is shown from stem Leydig cells to adult Leydig cells including the perivascular and peritubular cell layers in the human testis. EMT has also been associated with risk of tumor formation, but in a recent study using the same culture procedure no changes in DNA methylation, indicative of tumorigenesis, were observed [ 35 ]. In addition, in an allogenic transplantation study using mouse in vitro propagated spermatogonia where the exact same culture medium was used as in our current study, no increased risk of cancer was reported in long-term followed transplanted mice compared to control [ 65 ].…”

“…Protocols for the procurement, storage, isolation and culturing of mixed primary testicular cells (PTC) from cryopreserved human testicular biopsies were identical to and are described extensively in a previous publication [ 35 ]. In short, single cell suspensions were isolated from cryopreserved testicular biopsies using a two-step enzymatic tissue digestion step, followed by overnight differential plating in MEM medium to remove adherent somatic cells from the non-adherent germ cell fraction [ 73 ].…”

CNV Hotspots in Testicular Seminoma Tissue and Seminal Plasma

“…Strujik et al investigated CNVs in SE and reported that no CNV hotspot in SE was detected [ 37 ]. However, we detected CNV gain of all analysed genes in SE tissue.…”

Fathering a child after childhood cancer treatments

“…The main concern is that in vitro manipulation could affect the (epi-) genetic integrity of these cells and induce a carcinogenic phenotype or adverse health effects on the offspring. Throughout the years, several research groups have investigated possible chromosomal aberrations [18,19], and genetic or epigenetic instability of the SSC cultures in animal studies [18,[20][21][22] and human in vitro models [19,23]. Overall, the chromosomal integrity was conserved [18,19,23] as well as genomic stability [19,22].…”